Identification of Genetic Modifiers of TDP-43: Inflammatory Activation of Astrocytes for Neuroinflammation

Transactive response DNA-binding protein 43 (TDP-43) is a ubiquitously expressed DNA/RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 has been implicated in numerous aspects of the mRNA life cycle, as well as in cell toxicity and neuroinflammation. In this study, we used the toxicity of the TDP-43 expression in Saccharomyces cerevisiae as an assay to identify TDP-43 genetic interactions. Specifically, we transformed human TDP-43 cDNAs of wild-type or disease-associated mutants (M337V and Q331K) en masse into 4,653 homozygous diploid yeast deletion mutants and then used next-generation sequencing readouts of growth to identify yeast toxicity modifiers. Genetic interaction analysis provided a global view of TDP-43 pathways, some of which are known to be involved in cellular metabolic processes. Selected putative loci with the potential of genetic interactions with TDP-43 were assessed for associations with neurotoxicity and inflammatory activation of astrocytes. The pharmacological inhibition of succinate dehydrogenase flavoprotein subunit A (SDHA) and voltage-dependent anion-selective channel 3 (VDAC3) suppressed TDP-43-induced expression of proinflammatory cytokines in astrocytes, indicating the critical roles played by SDHA and VDAC3 in TDP-43 pathways during inflammatory activation of astrocytes and neuroinflammation. Thus, the findings of our TDP-43 genetic interaction screen provide a global landscape of TDP-43 pathways and may help improve our understanding of the roles of glia and neuroinflammation in ALS and FTD pathogenesis.

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Systematic characterization and genetic interaction analysis of adhesins in Candida albicans virulence

10.1101/2020.10.22.350991 ◽

2020 ◽

Author(s):

Sierra Rosiana ◽

Liyang Zhang ◽

Grace H. Kim ◽

Alexey V. Revtovich ◽

Arjun Sukumaran ◽

...

Keyword(s):

Candida Albicans ◽

Genetic Interaction ◽

Interaction Analysis ◽

Genetic Interactions ◽

Fungal Virulence ◽

C Elegans ◽

Adhesin Proteins ◽

Insight Into

AbstractCandida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans’ ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, or in every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.SummaryCandida albicans is a human fungal pathogen and cause of life-threatening systemic infections. Cell surface-associated adhesins play a central role in this pathogen’s ability to establish infection. Here, we provide a comprehensive analysis of adhesin factors, and their role in fungal virulence. Exploiting a high-throughput workflow, we screened an adhesin mutant library using C. elegans as a simple model host, and identified mutants and genetic interactions involved in virulence. We found that adhesin mutants are impaired in in vitro pathogenicity, irrespective of their virulence. Together, this work provides new insight into the role of adhesin factors in mediating fungal virulence.

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Comprehensive genetic analysis of adhesin proteins and their role in virulence ofCandida albicans

Genetics ◽

10.1093/genetics/iyab003 ◽

2021 ◽

Vol 217 (2) ◽

Author(s):

Sierra Rosiana ◽

Liyang Zhang ◽

Grace H Kim ◽

Alexey V Revtovich ◽

Deeva Uthayakumar ◽

...

Keyword(s):

Genetic Interaction ◽

Interaction Analysis ◽

Human Microbiome ◽

Opportunistic Pathogen ◽

Genetic Interactions ◽

C Elegans ◽

Cellular Morphogenesis ◽

Adhesin Proteins

AbstractCandida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans’ ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, and every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this study yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.

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Aberrant DJ-1 expression underlies L-type calcium channel hypoactivity in tuberous sclerosis complex and Alzheimer’s disease

10.1101/2020.08.26.267260 ◽

2020 ◽

Author(s):

Farr Niere ◽

Luisa P. Cacheaux ◽

Ayse Uneri ◽

Sanjeev Namjoshi ◽

Cameron Reynoldson ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Tuberous Sclerosis ◽

Neurological Disorders ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Cellular Mechanisms ◽

Mtorc1 Signaling ◽

Voltage Dependent

AbstractL-type voltage-dependent Ca2+ channels (L-VDCC) integrate synaptic signals to facilitate a plethora of cellular mechanisms. L-VDCC dysfunction is implicated in several neurological and psychiatric diseases. Despite their importance, signals upstream of L-VDCC activity that regulate their channel density, however, are poorly defined. In disease models with overactive mammalian target of rapamycin complex 1 (mTORC1) signaling (or mTORopathies), including tuberous sclerosis (TS) and Alzheimer’s disease (AD), we report a novel mechanism downstream of mTORC1 signaling that results in a deficit in dendritic L-VDCC activity. Deficits in L-VDCC activity are associated with increased expression of the mTORC1-regulated RNA-binding protein DJ-1. DJ-1 binds the mRNA coding the auxiliary Ca2+ channel subunit α2δ2 responsible for shuttling L-VDCC to the membrane and represses its expression. Moreover, this novel DJ-1/α2δ2/L-VDCC pathway is disrupted in human AD and preclinical models of AD and TS. Our discovery that DJ-1 directs L-VDCC activity and L-VDCC-associated protein α2δ2 at the synapse suggests that DJ-1/α2δ2/L-VDCC is a common, fundamental pathway disrupted in TS and AD that can be targeted in clinical mTORopathies.Significance StatementMany neurological disorders share symptoms, despite disparity among diseases. Treatments are prescribed based on diagnosis rather than individual symptoms. While only treating symptoms may obscure the disease, mechanism-based drug development allows the two approaches to converge. Hub proteins, those that coordinate the expression of proteins that mediate specific cellular functions, may be dysregulated across a broad range of disorders. Herein, we show that the RNA-binding protein DJ-1 controls the activity of L-type voltage-dependent calcium channels (L-VDCC), via the expression of its auxiliary subunit alpha2delta2 (α2δ2). Importantly, we demonstrate that this novel DJ-1/α2δ2/L-VDCC pathway is commonly disrupted among neurological disorders, namely Alzheimer’s disease (AD) and Tuberous Sclerosis (TS). Collectively, these data rationalize mechanism-based drug therapy to treat disease.

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Molecular basis for the differential interaction of plant mitochondrial VDAC proteins with tRNAs

Nucleic Acids Research ◽

10.1093/nar/gku728 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9937-9948 ◽

Cited By ~ 18

Author(s):

Thalia Salinas ◽

Samira El Farouk-Ameqrane ◽

Elodie Ubrig ◽

Claude Sauter ◽

Anne-Marie Duchêne ◽

...

Keyword(s):

Amino Acids ◽

Nucleic Acids ◽

Rna Binding ◽

Mitochondrial Outer Membrane ◽

Differential Interaction ◽

Voltage Dependent ◽

Selective Channel ◽

Trna Translocation ◽

Α Helix ◽

Trna Binding

Abstract In plants, the voltage-dependent anion-selective channel (VDAC) is a major component of a pathway involved in transfer RNA (tRNA) translocation through the mitochondrial outer membrane. However, the way in which VDAC proteins interact with tRNAs is still unknown. Potato mitochondria contain two major mitochondrial VDAC proteins, VDAC34 and VDAC36. These two proteins, composed of a N-terminal α-helix and of 19 β-strands forming a β-barrel structure, share 75% sequence identity. Here, using both northwestern and gel shift experiments, we report that these two proteins interact differentially with nucleic acids. VDAC34 binds more efficiently with tRNAs or other nucleic acids than VDAC36. To further identify specific features and critical amino acids required for tRNA binding, 21 VDAC34 mutants were constructed and analyzed by northwestern. This allowed us to show that the β-barrel structure of VDAC34 and the first 50 amino acids that contain the α-helix are essential for RNA binding. Altogether the work shows that during evolution, plant mitochondrial VDAC proteins have diverged so as to interact differentially with nucleic acids, and this may reflect their involvement in various specialized biological functions.

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A Genetic Screen Links the Disease-Associated Nab2 RNA-Binding Protein to the Planar Cell Polarity Pathway in Drosophila melanogaster

10.1101/2019.12.23.887257 ◽

2019 ◽

Author(s):

Wei-Hsuan Lee ◽

Edwin Corgiat ◽

J. Christopher Rounds ◽

Zenyth Shepherd ◽

Anita H. Corbett ◽

...

Keyword(s):

Cell Polarity ◽

Actin Polymerization ◽

Planar Cell Polarity ◽

Rna Binding ◽

Fragile X ◽

Binding Protein ◽

Rna Binding Protein ◽

Genetic Interactions ◽

Structural Defects ◽

Loss Of Function

ABSTRACTMutations in the gene encoding the ubiquitously expressed RNA-binding protein ZC3H14 result in a non-syndromic form of autosomal recessive intellectual disability. Studies in Drosophila have defined roles for the ZC3H14 ortholog, Nab2 (aka Drosophila Nab2 or dNab2), in axon guidance and memory due in part to interaction with a second RNA-binding protein, the fly Fragile X homolog Fmr1, and coregulation of shared Nab2-Fmr1 target mRNAs. Despite these advances, neurodevelopmental pathways regulated by Nab2 remain poorly defined. Structural defects in Nab2 null brains resemble defects observed upon disruption of the planar cell polarity (PCP) pathway, which regulates planar orientation of static and motile cells. A kinked bristle phenotype in surviving Nab2 mutant adults additionally suggests a defect in F-actin polymerization and bundling, which is also a PCP-regulated processes. To test for Nab2-PCP genetic interactions, a collection of PCP loss-of-function alleles was screened for modification of a rough-eye phenotype produced by Nab2 overexpression in the eye (GMR-Nab2) and subsequently for modification of Nab2 null phenotypes. Multiple PCP alleles dominantly modify GMR-Nab2 eye roughening and a subset of these alleles also rescue low survival and thoracic bristle kinking in Nab2 zygotic nulls. Moreover, alleles of two X-linked PCP factors, dishevelled (dsh) and β amyloid protein precursor-like (Appl), rescue GMR-Nab2 eye roughening in male progeny derived from hemizygous dsh or Appl mutant fathers, suggesting an additional effect inherited through the male germline. These findings demonstrate a consistent pattern of Nab2-PCP genetic interactions that suggest molecular links between Nab2 and the PCP pathway in the developing eye, wing and germline.

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A Genetic Screen Links the Disease-Associated Nab2 RNA-Binding Protein to the Planar Cell Polarity Pathway in Drosophila melanogaster

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401637 ◽

2020 ◽

Vol 10 (10) ◽

pp. 3575-3583 ◽

Cited By ~ 1

Author(s):

Wei-Hsuan Lee ◽

Edwin Corgiat ◽

J. Christopher Rounds ◽

Zenyth Shepherd ◽

Anita H. Corbett ◽

...

Keyword(s):

Cell Polarity ◽

Actin Polymerization ◽

Planar Cell Polarity ◽

Rna Binding ◽

Fragile X ◽

Binding Protein ◽

Rna Binding Protein ◽

Genetic Interactions ◽

Gene Encoding ◽

Link Type

Mutations in the gene encoding the ubiquitously expressed RNA-binding protein ZC3H14 result in a non-syndromic form of autosomal recessive intellectual disability in humans. Studies in Drosophila have defined roles for the ZC3H14 ortholog, Nab2 (aka Drosophila Nab2 or dNab2), in axon guidance and memory due in part to interaction with a second RNA-binding protein, the fly Fragile X homolog Fmr1, and coregulation of shared Nab2-Fmr1 target mRNAs. Despite these advances, neurodevelopmental mechanisms that underlie defective axonogenesis in Nab2 mutants remain undefined. Nab2 null phenotypes in the brain mushroom bodies (MBs) resemble defects caused by alleles that disrupt the planar cell polarity (PCP) pathway, which regulates planar orientation of static and motile cells via a non-canonical arm of the Wnt/Wg pathway. A kinked bristle phenotype in surviving Nab2 mutant adults additionally suggests a defect in F-actin polymerization and bundling, a PCP-regulated processes. To test for Nab2-PCP genetic interactions, a collection of PCP mutant alleles was screened for modification of a rough-eye phenotype produced by Nab2 overexpression in the eye (GMR>Nab2) and, subsequently, for modification of a viability defect among Nab2 nulls. Multiple PCP alleles dominantly modify GMR>Nab2 eye roughening and a subset rescue low survival and thoracic bristle kinking in Nab2 zygotic nulls. Collectively, these genetic interactions identify the PCP pathway as a potential target of the Nab2 RNA-binding protein in developing eye and wing tissues and suggest that altered PCP signaling could contribute to neurological defects that result from loss of Drosophila Nab2 or its vertebrate ortholog ZC3H14.

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